Crystallization Studies of the TraF Protein from the F Plasmid of Escherichia coli

Crystallization of suitable crystals for diffraction analysis is a major hurdle in structure-function studies of proteins using X-ray crystallography. This is especially true when the protein may be generally hydrophobic, membrane associated or contain regions of increased flexibility. The objective of the research outlined in this thesis was to determine a more amenable set of crystallization conditions for the protein TraF from the F plasmid for X-ray diffraction and structure solution. The F plasmid of Escherichia coli utilizes a conjugative type IV secretion system (T4SS) by which the transmission of genes in bacteria occurs contributing to the current issue of antibiotic resistance. The TraF protein of this system is a periplasmic protein with a dynamic hydrophobic N-terminal tail which interacts directly with TraH and a C-terminal thioredoxin-like domain. TraF is hypothesized to chaperone and interact with several other proteins of this system. Although its exact function remains unclear, TraF is essential in in the F plasmids T4SS as a lack of function abolishes gene transfer through this system. Modification of upstream conditions, including expression and purification of a GST-tagged variant of TraF, as well as modification of buffer conditions including the detergent NP-40 allowed for an increased solubility and concentration of the protein for crystallization trials. The traditional screening process with GST-TraF resulted in two conditions that showed birefringent structures which did not yield any better results when optimized. Optimization of the original reported TraF purification and crystallization conditions did double the protein concentration, however resulted in a lack of miscibility between the protein stock and the reservoir solution during crystallization. Furthermore, a head-to-head crystallization screening was conducted incorporating 5-amino-2,4,6-triiodoisophthalic (I3C) in the crystallization drop to advance structure solution using experimental phasing methods. The head-to-head experiment led to multiple crystallization conditions that should be optimized and further assessed while validating the novel process of incorporating I3C in the screen formation process.